Antimicrobial potential of quaternary phosphonium salt compounds: a review.

Given that mitochondrial dysregulation is a biomarker of many cancers, cationic quaternary phosphonium salt (QPS) conjugation is a widely utilized strategy for anticancer drug design. QPS-conjugated compounds exhibit greater cell permeation and accumulation in negatively charged mitochondria, and thus, show enhanced activity. Phylogenetic similarities between mitochondria and bacteria have provided a rationale for exploring the antibacterial properties of mitochondria-targeted compounds. Additionally, due to the importance of mitochondria in the survival of pathogenic microbes, including fungi and parasites, this strategy can be extended to these organisms as well. This review examines recent literature on the antimicrobial activities of various QPS-conjugated compounds and provides future directions for exploring the medicinal chemistry of these compounds.

The expanding repertoire of covalent warheads for drug discovery.

The reactive functionalities of drugs that engage in covalent interactions with the enzyme/receptor residue in either a reversible or an irreversible manner are called 'warheads'. Covalent warheads that were previously neglected because of safety concerns have recently gained center stage as a result of their various advantages over noncovalent drugs, including increased selectivity, increased residence time, and higher potency. With the approval of several covalent inhibitors over the past decade, research in this area has accelerated. Various strategies are being continuously developed to tune the characteristics of warheads to improve their potency and mitigate toxicity. Here, we review research progress in warhead discovery over the past 5 years to provide valuable insights for future drug discovery.

Elemental exchange: Bioisosteric replacement of phosphorus by boron in drug design.

Continuous efforts are being directed toward the employment of boron in drug design due to its advantages and unique characteristics including a plethora of target engagement modes, lower metabolism, and synthetic accessibility, among others. Phosphates are components of multiple drug molecules as well as clinical candidates, since they play a vital role in various biochemical functions, being components of nucleotides, energy currency- ATP as well as several enzyme cofactors. This review discusses the unique chemistry of boron functionalities as phosphate bioisosteres - "the boron-phosphorus elemental exchange strategy" as well as the superiority of boron groups over other commonly employed phosphate bioisosteres. Boron phosphate-mimetics have been utilized for the development of enzyme inhibitors as well as novel borononucleotides. Both the boron functionalities described in this review-boronic acids and benzoxaboroles-contain a boron connected to two oxygens and one carbon atom. The boron atom of these functional groups coordinates with a water molecule in the enzyme site forming a tetrahedral molecule which mimics the phosphate structure. Although boron phosphate-mimetic molecules - FDA-approved Crisaborole and phase II/III clinical candidate Acoziborole are products of the boron-phosphorus bioisosteric elemental exchange strategy, this technique is still in its infancy. The review aims to promote the use of this strategy in future medicinal chemistry projects.

Mitochondriotropic Derivative of Ethyl Ferulate, a Dietary Phenylpropanoid, Exhibits Enhanced Cytotoxicity in Cancer Cells via Mitochondrial Superoxide-Mediated Activation of JNK and AKT Signalling.

Specific targeting of anti-cancer drugs to mitochondria is an emerging strategy to enhance cancer cell killing whilst simultaneously overcoming the problem of drug resistance, low bioavailability and limited clinical success of natural products. We have synthesized a mitochondria targeted derivative of Ethyl Ferulate (EF, a naturally occurring ester of ferulic acid), by conjugating it with triphenylphosphonium ion and compared its cytotoxicity with the parent molecule. Mito-Ethyl Ferulate (M-EF) was found to be more potent than EF (~ 400-fold) in inhibiting the growth of A549 and MCF-7 cells and suppressing the clonogenic potential of A549 cells. Notably, M-EF did not induce any cytotoxicity in normal cells (mouse normal fibroblast cells) up to a concentration of 25 µM. Furthermore, M-EF treatment induced significantly higher cell death in MCF-7 and A549 cells, as compared to EF via induction of apoptosis. M-EF treatment increased mitochondrial superoxide production and induced mitochondrial DNA damage and phosphorylation of JNK and AKT in A549 cells. Furthermore, M-EF induced increase in mitochondrial superoxide production and cytotoxicity was attenuated on pre-treatment with mitochondria-targeted antioxidant (mitoTEMPO) indicating the involvement of mitochondrial ROS in the cytotoxic effects of M-EF. Finally, in silico prediction revealed putative mitochondrial targets of M-EF which are known to regulate mitochondrial ROS and cell viability. In conclusion, the improved cytotoxic efficacy of M-EF exemplifies the use of mitochondria-specific drug delivery in future development of natural product based mitochondrial pharmacology.

Identification of bicyclic compounds that act as dual inhibitors of Bcl-2 and Mcl-1.

Elevated expression of anti-apoptotic proteins, such as Bcl-2 and Mcl-1 contributes to poor prognosis and resistance to current treatment modalities in multiple cancers. Here, we report the design, synthesis and characterization of benzimidazole chalcone and flavonoid scaffold-derived bicyclic compounds targeting both Bcl-2 and Mcl-1 by optimizing the structural differences in the binding sites of both these proteins. Initial docking screen of Bcl-2 and Mcl-1 with pro-apoptotic protein Bim revealed possible hits with optimal binding energies. All the optimized bicyclic compounds were screened for their in vitro cytotoxic activity against two oral cancer cell lines (AW8507 and AW13516) which express high levels of Bcl-2 and Mcl-1. Compound 4d from the benzimidazole chalcone series and compound 6d from the flavonoid series exhibited significant cytotoxic activity (IC50 7.12 µM and 17.18 µM, respectively) against AW13516 cell line. Time Resolved-Fluorescence Resonance Energy Transfer (TR-FRET) analysis further demonstrated that compound 4d and compound 6d could effectively inhibit the Bcl-2 and Mcl-1 proteins by displacing their BH3 binding partners. Both compounds exhibited potent activation of canonical pathway of apoptosis evident from appearance of cleaved Caspase-3 and PARP. Further, treatment of oral cancer cells with the inhibitors induced dissociation of the BH3 only protein Bim from Mcl-1 and Bak from Bcl-2 but failed to release Bax from Bcl-xL thereby confirming the nature of compounds as BH3-mimetics selectively targeting Bcl-2 and Mcl-1. Our study thus identifies bicyclic compounds as promising candidates for anti-apoptotic Bcl-2/Mcl-1 dual inhibitors with a potential for further development.

Role of calcium dysregulation in Alzheimer's disease and its therapeutic implications.

The increasing incidence of Alzheimer's disease (AD) coupled with the lack of therapeutics to address the underlying pathology of the disease has necessitated the need for exploring newer targets. Calcium dysregulation represents a relatively newer target associated with AD. Ca+2 serves as an important cellular messenger in neurons. The concentration of the Ca+2 ion needs to be regulated at optimal concentrations intracellularly for normal functioning of the neurons. This is achieved with the help of mitochondria, endoplasmic reticulum, and neuronal plasma membrane channel proteins. Disruption in normal calcium homeostasis can induce formation of amyloid beta plaques, accumulation of neurofibrillary tangles, and dysfunction of synaptic plasticity, which in turn can affect calcium homeostasis further, thus forming a vicious cycle. Hence, understanding calcium dysregulation can prove to be a key to develop newer therapeutics. This review provides detailed account of physiology of calcium homeostasis and its dysregulation associated with AD. Further, with an understanding of various receptors and organelles involved in these pathways, the review also discusses various calcium channel blockers explored in AD hand in hand with some multitarget molecules addressing calcium as one of the targets.

Antituberculosis activity of polyphenols of Areca catechu.

Background: Polyphenols have been studied for their potential involvement in the prevention of various chronic diseases as well as for their antimicrobial potential. The crude extracts of arecanut have been reported to have antiinfective properties. We aimed to explore the endosperm of Areca catechu (arecanut) for the extraction of polyphenol components and to study the antituberculosis activity of these polyphenol against Mycobacterium tuberculosis H37Rv. Method: A comparative extraction was performed using microwave and Soxlet apparatus. High performance liquid chromatography (HPLC) technique was used for the estimation of the extracted polyphenols. The minimum inhibitory concentration (MIC) values against M.tuberculosis H37Rv stain, Staphylococcus aureus and Escherichia coli were estimated by resazurin microtiter assay. Results: There was a 11-fold increase in the total phenolic content by microwave assisted extraction compared to the Soxhlet extraction. The powdered extract was found to be active with MIC value of 0.975 ± 0.02 µg/mL. Fractionation and HPLC-based estimation of the extract revealed catechin, epicatechin, and epigallocatechin gallate to be the polyphenol components in the ethanol fraction. Conclusions: The bioactivity of these polyphenols confirmed their presence and complementary effect in the extract form. Because the toxic alkaloid arecoline, known to be present in arecanut, did not show any activity individually, the bioactivity of the extract was attributed to the nontoxic polyphenols present. This extract also showed selective inhibition of M. tuberculosis over other gram positive and gram-negative bacteria, thereby establishing that arecanut is an exploitable selective source of polyphenols acting against M. tuberculosis.

Mutations in SARS-CoV-2 nsp7 and nsp8 proteins and their predicted impact on replication/transcription complex structure.

Severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) RNA-dependent RNA polymerase (RdRp) has been identified to be a mutation hot spot, with the P323L mutation being commonly observed in viral genomes isolated from North America. RdRp forms a complex with nonstructural proteins nsp7 and nsp8 to form the minimal replication/transcription machinery required for genome replication. As mutations in RdRp may affect formation of the RdRp-nsp7-nsp8 supercomplex, we analyzed viral genomes to identify mutations in nsp7 and nsp8 protein sequences. Based on in silico analysis of predicted structures of the supercomplex comprising of native and mutated proteins, we demonstrate that specific mutations in nsp7 and nsp8 proteins may have a role in stabilization of the replication/transcription complex.

Exploring the potential of pyrazoline containing molecules as Aß aggregation inhibitors in Alzheimer's disease.

Objectives Alzheimer's disease (AD) is a chronic and progressive neurodegenerative disease in which one of the most prominent pathological features is accumulation of amyloid (Aß) plaques. This occurs due to the process of aggregation from monomeric to polymeric forms of Aß peptide and thus represents one of the attractive targets to treat AD. Methods After initial evaluation of a set of molecules containing N-acetylpyrazoline moiety flanked by aromatic rings on both sides as Aß aggregation inhibitors, the most potent molecules were further investigated for mechanistic insights. These were carried out by employing techniques such as circular dichroism (CD) spectroscopy, transmission electron microscopy (TEM), in vitro PAMPA-BBB (Blood-Brain Barrier) assay and cytotoxicity evaluation. Results Two molecules among the exploratory set displayed Aß aggregation inhibition comparable to standard curcumin. Among the follow-up molecules, several molecules displayed more inhibition than curcumin. These molecules displayed good inhibitory activity even at lower concentrations. CD and TEM confirmed the mechanism of Aß aggregation. These molecules were found to alleviate Aß induced cytotoxicity. BBB penetration studies highlighted the potential of these molecules to reach central nervous system (CNS). Conclusions Thus, several promising Aß-aggregation inhibitors were obtained as a result of this study.

3-(5-Nitrofuran-2-yl)prop-2-en-1-one Derivatives, with Potent Antituberculosis Activity, Inhibit A Novel Therapeutic Target, Arylamine N-acetyltransferase, in Mycobacteria.

In this study, the inhibitory potential of 3-(5-nitrofuran-2-yl)prop-2-en-1-one derivatives was evaluated against a panel of bacteria, as well as mammalian cell lines to determine their therapeutic index. In addition, we investigated the mechanism of antibiotic action of the derivatives to identify their therapeutic target. We discovered compound 2 to be an extremely potent inhibitor of Mycobacterium tuberculosis H37Rv growth (MIC: 0.031 mg/L) in vitro, performing better than the currently used first-line antituberculosis drugs such as isoniazid, rifampicin, ethambutol, and pretomanid in vitro. Furthermore, compound 3 was equipotent to pretomanid against a multidrug-resistant M. tuberculosis clinical isolate. The derivatives were selective and bactericidal towards slow-growing mycobacteria. They showed low cytotoxicity towards murine RAW 264.7 and human THP-1 cell lines, with high selectivity indices. Compound 1 effectively eliminated the intracellular mycobacteria in a mycobacteria-infected macrophage model. The derivatives were assessed for their potential to inhibit mycobacterial arylamine N-acetyltransferase (NAT) and were identified as good inhibitors of recombinant mycobacterial NAT, a novel target essential for the intracellular survival of M. tuberculosis. This study provided hits for designing new potent and selective antituberculosis leads, having mycobacterial NAT inhibition as their possible endogenous mechanisms of action.

Tackling SARS-CoV-2: proposed targets and repurposed drugs.

The SARS-CoV-2 pandemic, declared as a global health emergency by the WHO in February 2020, has currently infected more than 6 million people with fatalities near 371,000 and increasing exponentially, in absence of vaccines and drugs. The pathogenesis of SARS-CoV-2 is still being elucidated. Identifying potential targets and repurposing drugs as therapeutic options is the need of the hour. In this review, we focus on potential druggable targets and suitable therapeutics, currently being explored in clinical trials, to treat SARS-CoV-2 infection. A brief understanding of the complex interactions of both viral as well as host targets, and the possible repurposed drug candidates are described with an emphasis on understanding the mechanisms at the molecular level.

In silico modeling of functionalized poly(methylvinyl ether/maleic acid) for controlled drug release in the ocular milieu.

Controlling structurally defined properties of drug-bound macromolecules such as surface adhesion and interaction with endogenous proteins in the surrounding environment using prior data from computer-assisted simulation can be of great use in designing controlled release macromolecular therapeutic systems. In this paper, we describe experimental correlation of real-time properties of a polymer with pendant drug molecules, with predicted values obtained from studying in silico molecular interactions of this polymer with ocular surface proteins (mucin) for formulating an ophthalmic in situ gel. Mucoretention of the drug (norfloxacin) within the eye sac is closely associated with binding interactions occurring on the ocular surface, and covalent association of the drug with the mucoadhesive polymer, poly(methylvinyl ether/maleic acid), can largely reduce dosing frequency eliciting prolonged antibacterial action much required in treating conjunctival infections. The physicochemical properties and 3D conformation of the drug-polymer conjugate were predicted by computational studies. Molecular docking of the drug-polymer conjugate with ocular surface mucin (MUC-1) suggested that amino acid residues Arg1095, Asn1091, and Gln1070 of mucin are involved in hydrogen bonding with carboxyl residues in the polymer structure. The orientation of the drug-polymer conjugate in solution profoundly depends on the properties of the drug. The studies further reveal that molecular interactions of MUC-1 with the drug in the drug-polymer conjugate influence the binding orientation of the drug-polymer to mucin. Computationally predicted solvation energies revealed a significant difference in energy values between drug molecule alone (- 113.04 kcal/mol) and the drug-polymer (- 492.44 kcal/mol) suggesting higher aqueous solvation of the drug-polymer conjugate compared with less-soluble drug, and that interactions between polymer chains and ocular aqueous environment dictate the drug-polymer conjugate's free energy. Our results demonstrate the fabrication of a macromolecular therapeutic gel using drug-polymer with controlled release properties and mucoadhesion guided by information predicted from computational software. Notably, in silico studies reveal that even small variations in molecular composition, in this case, an antibacterial drug that contributes less than half of the entire molecular weight can considerably change the drug's presentation to the ocular environment. Graphical abstract Table of contents graphic.

Network topology of LMWG cross-linked xyloglucan hydrogels for embedding hydrophobic nanodroplets: mechanistic insight and molecular dynamics.

Indigenous polymers have functional implications in biomedicine due to the presence of an inherent favorable structural architecture that supports hydrogel formation. In this study, we present the molecular level characterization of xyloglucan hydrogels using experimental and molecular simulation methods. We studied supramolecular self-assembly of tamarind seed-derived xyloglucan induced by low molecular weight gelators to form dense networks and rationalized its capabilities as a multifunctional and multiresponsive matrix for holding hydrophobic nanometric oleic acid globules intact for extended periods, preventing coalescence triggered instability using computational methods and imaging. Computational studies using molecular dynamics simulations provided mechanistic evidences of molecular associations supported by strong hydrogen bonding interactions responsible for fundamental gel network formation. Real-time imaging studies revealed that the gel matrix immobilizes intact oleic acid globules within its framework preventing coalescence. Molecular dynamics studies further showed key interactions of xyloglucan with the surfactant polysorbate 60 involved in sustaining oleic acid globules within the gel matrix, which corroborate with FE-SEM results. This study is an interesting approach to understand how molecular level associations in xyloglucan-based hydrogels can control and preserve nanosized hydrophobic oils imparting tenability and long-term droplet stability. The study also brings new insights into the conformational flexibility of xyloglucan around molecules with favorable and unfavorable interactive chemistries. Graphical abstract .

Exploration of 5-(5-nitrothiophen-2-yl)-4,5-dihydro-1H-pyrazoles as selective, multitargeted antimycobacterial agents.

We report the biological evaluation of 5-(5-nitrothiophen-2-yl)-4,5-dihydro-1H-pyrazole derivatives against bacteria, eukaryotic cell lines and the assessment of their mechanisms of action to determine their prospects of being developed into potent antituberculosis agents. The compounds were evaluated for their antibacterial property against Mycobacterium tuberculosis H37Rv, multidrug-resistant M. tuberculosis, Mycobacterium bovis BCG, Mycobacterium aurum, Escherichia coli, and Staphylococcus aureus using high-throughput spot-culture growth inhibition assay. They were found to be selective toward slow-growing mycobacteria and Gram-positive bacteria. In M. bovis BCG, they exhibited a bactericidal mode of action. Cytotoxicity was assessed in human THP-1 and murine RAW 264.7 cell lines, and the compounds showed a lower cytotoxicity potential when compared with their antibacterial activity. They were found to be excellent whole-cell efflux pump inhibitors of the mycobacterial surrogate M. aurum, performing better than known efflux pump inhibitor verapamil. The 5-nitrothiophene moiety was identified for the first time as a prospective inhibitor scaffold of mycobacterial arylamine N-acetyltransferase enzyme, which is the key enzyme in metabolizing isoniazid, a first-line antituberculosis drug. The two aforementioned findings make the compounds potential hits in the development of adjunctive tuberculosis therapy.

Targeting cancer's Achilles' heel: role of BCL-2 inhibitors in cellular senescence and apoptosis.

Members of the antiapoptotic BCL-2 proteins are involved in tumor growth, progression and survival, and are also responsible for chemoresistance to conventional anticancer agents. Early efforts to target these proteins yielded some active compounds; however, newer methodologies involving structure-based drug design, Nuclear Magnetic Resonance (NMR)-based screening and fragment-based screening yielded more potent compounds. Discovery of specific as well as nonspecific inhibitors of this class of proteins has resulted in great advances in targeted chemotherapy and decrease in chemoresistance. Here, we review the history and current progress in direct as well as selective targeting of the BCL-2 proteins for anticancer therapy.

Synthesis and in-vivo taste assessment of meloxicam pivalate.

Meloxicam (MX), a nonsteroidal anti-inflammatory drug, widely used to treat arthritis, has a very bitter taste. Chemical modification of the bitter functionality was achieved by synthesis of a prodrug, meloxicam pivalate (MXP). Taste improvement was evaluated using single bottle-test rat model. It was found that palatability of MXP solution improved significantly as compared to MX.

Synthesis and mycobacterial evaluation of 5-substituted-6-acetyl-2-amino-7-methyl-5,8-dihydropyrido-[2,3-d]pyrimidin-4(3H)-one derivatives.

5-Substituted-6-acetyl-2-amino-7-methyl-5,8-dihydropyrido[2,3-d]pyrimidin-4(3H)-one derivatives were synthesized and evaluated against Mycobacterium tuberculosis H37Rv, Mycobacterium aurum, Escherichia coli, and Staphylococcus aureus as well as a human monocyte-derived macrophage (THP-1), and murine macrophage (RAW 264.7) cell lines to assess their antibacterial and cytotoxic potential, respectively. The compounds showed activity in the range of 1.95-125 µg/ml against M. tuberculosis but showed no activity against M. aurum, E. coli, and S. aureus, indicating selectivity towards slow-growing mycobacterial pathogens. The compounds exhibited very low to no cytotoxicity up to 500 µg/ml concentration against eukaryotic cell lines. The most potent molecule, 2l, showed a minimum inhibitory concentration of 1.95 µg/ml against M. tuberculosis H37Rv and a selectivity index of >250 against both the eukaryotic cell lines. Furthermore, 2l showed moderate inhibition of whole-cell mycobacterial drug-efflux pumps when compared to verapamil, a known potent inhibitor of efflux pumps. Thus, derivative 2l was identified as an antituberculosis hit molecule, which could be used to yield more potent lead molecules.

Oxadiazole scaffolds in anti-tuberculosis drug discovery.

With the increasing number of cases of latent and drug resistant tuberculosis, there is an urgent need to develop new, potent molecules capable of combating this deadly disease. Molecules containing oxadiazoles are one such class that could be considered to fulfil this need. Oxadiazole regioisomers have been explored in drug discovery programs for their ability to act as effective linkers and also as pharmacophoric features. Oxadiazoles can act as bioisosteric replacements for the hydrazide moiety which can be found in first line anti-TB drugs, and some have been also reported to interact with newer anti-TB targets. In this context, the present review describes the potential of oxadiazoles as antituberculosis agents.

Evaluation of Phytopolyphenols for their gp120-CD4 Binding Inhibitory Properties by In Silico Molecular Modelling & In Vitro Cell Line Studies.

BACKGROUND: Lack of effective early-stage HIV-1 inhibitor instigated the need for screening of novel gp120-CD4 binding inhibitor. Polyphenols, a secondary metabolite derived from natural sources are reported to have broad spectrum HIV-1 inhibitory activity. However, the gp120-CD4 binding inhibitory activity of polyphenols has not been analysed in silico yet. OBJECTIVES: To establish the usage of phytopolyphenols (Theaflavin, Epigallocatechin (EGCG), Ellagic acid and Gallic acid) as early stage HIV-1 inhibitor by investigating their binding mode in reported homology of gp120-CD4 receptor complex using in silico screening studies and in vitro cell line studies. METHODS: The in silico molecular docking and molecular simulation studies were performed using Schrödinger 2013-2 suite installed on Fujitsu Celsius Workstation. The in vitro cell line studies were performed in the TZM-bl cell line using MTT assay and ß-galactosidase assay. RESULTS: The results of molecular docking indicated that Theaflavin and EGCG exhibited high XP dock score with binding pose exhibiting Van der Waals interaction and hydrophobic interaction at the deeper site in the Phe43 cavity with Asp368 and Trp427. Both Theaflavin and EGCG form a stable complex with the prepared HIV-1 receptor and their binding mode interaction is within the vicinity 4 Å. Further, in vitro cell line studies also confirmed that Theaflavin (SI = 252) and EGCG (SI = 138) exert better HIV-1 inhibitory activity as compared to Ellagic acid (SI = 30) and Gallic acid (SI = 34). CONCLUSION: The results elucidate a possible binding mode of phytopolyphenols, which pinpoints their plausible mechanism and directs their usage as early stage HIV-1 inhibitor.

Tetrahydrocurcumin-loaded vaginal nanomicrobicide for prophylaxis of HIV/AIDS: in silico study, formulation development, and in vitro evaluation.

A vaginal microbicide is a front-line women-dependent approach and an alternative to a condom for prevention of unprotected sexual intercourse-associated HIV. The microbicide research is still in its infancy with several products in the clinical studies being reported to have good efficacy, safe, but with poor adherence. One such molecule reported with an excellent efficacy when tested preclinically is curcumin, a natural polyphenol derived from Curcuma longa. Despite its potential HIV-1 inhibitory activity, it has intense yellow color staining properties, which would result in poor consumer compliance and adherence for vaginal application. To address this issue, tetrahydrocurcumin (THC), a colorless derivative of curcumin, was subjected to in silico screening (molecular docking and dynamics simulation studies) using homology model of gp120-CD4 binding. It was found that THC exhibited equivalent gp120-CD4 binding inhibitory activity as compared with curcumin due to its stable hydrophobic interactions with residues Asp368 and Trp427 deeper in the Phe43 cavity of CD4 receptor. Hence, it can be effectively used as a potential microbicide candidate. THC, a BCS Class II molecule exhibits poor solubility, spreadability, and intracellular uptake when used in the conventional form. Thus, it was decided to develop a lipid-based nanomicrobicide gel for delivery of THC. The developed THC-loaded o/w microemulsion gel was characterized for physicochemical properties (globule size, drug content, drug release, and permeation) and further used for in vitro cell line studies (cell viability, cellular uptake, and anti-HIV activity). The developed formulation was found to be stable with coitus-independent release profile and exhibited a rapid time-independent intracellular uptake. In addition, it exhibited a fourfold increase in efficacy as compared with conventional THC. Thus, the novel THC-loaded o/w microemulsion gel exhibited the potential for prevention of HIV-1 infection associated with unprotected sexual intercourse.